Myeloid Translocation Genes (MTGs) were first discovered in Acute Myeloid Leukemia fusion proteins and have been found to be a family of three highly homologous transcriptional co-repressors that are widely expressed in normal physiology. In order to better understand the effects of the leukemogenic fusion proteins, we are investigating the normal functions of MTGs through knock-out mouse models;this proposal will focus on the Mtg16-null model. Among other hematopoietic phenotypes, Mtg16-null mice have a marked decrease in total thymocytes due to an unknown mechanism. The research proposed here thus has two aims. First, we aim to characterize the defects in thymocyte development in Mtg16-null mice. To achieve this end, we will first use thymocyte cell surface markers and flow cytometry to determine differences between the maturation of Mtg16-null and wild-type thymocytes. We will follow this with studies such as BrdU labeling and Annexin V staining to define the nature of the observed differences. Microarray analysis of the changes in gene expression that result from Mtg16 deletion will give new insight into both the mechanisms underlying decreases in thymocyte number and Mtg16 targets and functions. In the second portion of this research, we will characterize CSL, the transcription factor of the Notch cascade, as a newly- identified transcription-factor binding partner for Mtg16. We hypothesize that this interaction provides a potential mechanism by which Mtg16 regulates thymocyte development. As MTGs are unable to bind DNA directly and instead rely on interactions with transcription factors to target their effects, we expect to show that by binding to CSL, Mtg16 regulates Notch signaling. Using co-immunoprecipitation, luciferase assays, and ChIP, we aim to identify the mechanism of interaction, the effects of this interaction, and some of the genes directly regulated by these complexes. We will also investigate the impact of misregulation of MTGs in fusion proteins on Notch signaling. This information will provide new insight into how the MTG family functions in lymphoid development and leukemogenesis. PUBLIC HEALTH RELEVANCE: Myeloid Translocation Genes (MTGs) are involved in mutations that produce Acute Myeloid Leukemia (AML) and by better understanding the normal functions of MTGs, we can better understand how they function in leukemia. We have identified a new role for MTG16, an MTG family member, in regulating gene expression during the development of the immune system. We are also investigating the role of MTG16 in regulating the Notch signaling cascade, a system critical for immune system development;this interaction may account for MTG16's effect on the immune system and the role of mutated MTGs in AML.